Radix isatidis Polysaccharides Inhibit Influenza a Virus and Influenza A Virus-Induced Inflammation via Suppression of Host TLR3 Signaling In Vitro

Abstract

Influenza remains one of the major epidemic diseases worldwide, and rapid virus replication and collateral lung tissue damage caused by excessive pro-inflammatory host immune cell responses lead to high mortality rates. Thus, novel therapeutic agents that control influenza A virus (IAV) propagation and attenuate excessive pro-inflammatory responses are needed. Polysaccharide extract from Radix isatidis, a traditional Chinese herbal medicine, exerted potent anti-IAV activity against human seasonal influenza viruses (H1N1 and H3N2) and avian influenza viruses (H6N2 and H9N2) in vitro. The polysaccharides also significantly reduced the expression of pro-inflammatory cytokines (IL-6) and chemokines (IP-10, MIG, and CCL-5) stimulated by A/PR/8/34 (H1N1) at a range of doses (7.5 mg/mL, 15 mg/mL, and 30 mg/mL); however, they were only effective against progeny virus at a high dose. Similar activity was detected against inflammation induced by avian influenza virus H9N2. The polysaccharides strongly inhibited the protein expression of TLR-3 induced by PR8, suggesting that they impair the upregulation of pro-inflammatory factors induced by IAV by inhibiting activation of the TLR-3 signaling pathway. The polysaccharide extract from Radix isatidis root therefore has the potential to be used as an adjunct to antiviral therapy for the treatment of IAV infection.

Keywords: Radix isatidis; TLR-3; anti-inflammatory; antiviral; polysaccharides.

Figure 1

Radix isatidis polysaccharides showed anti-inflammation activity in 16HBE cells infected with PR8/H1N1 virus. (A) Cell viability was evaluated as described in Materials and Methods and expressed as a percentage of the vehicle control. After mock-infection or infection with PR8 (MOI = 0.1 TCID₅₀/cell), 16HBE cells were treated with R. isatidis polysaccharides or 0.5% DMSO. Total RNA of the 16HBE cells at 6 h (B) or 24 h (C) was isolated, and RT-qPCR was performed. Samples were normalised to GAPDH as a control. The protein level of IP-10 was tested by ELISA (D), and the virus titer in the supernatant was tested by the CPE method (E). RIP: R. isatidis polysaccharides. Data are shown as the mean ± SD for three independent experiments. Statistical significance was evaluated using the Student’s t-test, relative to the mock-infected cells (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 2

Radix isatidis polysaccharides can inhibit cytokine expression induced by H9N2 avian influenza A virus in 16HBE cells. After being mock-infected or infected with H9N2 (MOI = 1), 16HBE cells were treated with R. isatidis polysaccharides or 0.5% DMSO. The protein levels of IP-10, IL-6, and CCL-5 were tested by ELISA. RIP: R. isatidis polysaccharides. Statistical significance was evaluated using the Student’s t-test, relative to the mock-infected cells (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 3

Radix isatidis polysaccharides can reduce the expression of TLR3 induced by PR8/H1N1 virus. 16HBE cells were infected with PR8/H1N1 virus (MOI = 1), then treated with R. isatidis polysaccharides. After 24 h, Western blotting was performed to assess the TLR3 protein level, and GAPDH protein was used as a control. RIP: R. isatidis polysaccharides. Statistical significance was evaluated using the Student’s t-test, relative to the mock-infected cells (* p < 0.05, ** p < 0.01).

Figure 4

Schematic diagram of the mechanism of suppression of influenza virus-induced inflammation by Radix isatidis polysaccharides.